We propose the first experimental analysis of the mechanisms by which transcription factors (TFs) evolved specificity for new DNA binding sites. The diversity and specificity of TFs allows organisms to precisely regulate cellular processes in development and physiology; modulation of TF action is also a critical means by which organisms evolve. But little is known about the mechanisms and dynamics by which TF's evolved their DNA specificities. Comparative studies of extant proteins have had limited success, because the causes of protein diversity occurred in the deep past, so historical approaches are required to distinguish them from the many other changes that have accrued since that time. Here we combine a powerful strategy for analyzing evolutionary mechanisms and processes-ancestral protein reconstruction-with advanced biophysical analysis and high-throughput screening of variant protein libraries to analyze the evolution of DNA specificity in the steroid hormone receptor (SR) protein family, a superb model of TF diversification. SRs play key roles in development, reproduction, homeostasis, cancer, and many diseases. The two classes of SRs-estrogen receptors on one hand and the receptors for androgens, progestagens, glucocorticoids, and mineralocorticoids (APGMRs) on the other-recognize different DNA binding sites. Preliminary data indicate that this diversity evolved via a sharp shift in DNA recognition that occurred between the ancestor of all steroid receptors (AncSR1) and the ancestor of the APGMRs (AncSR2). Our goals are to: 1) Dissect this evolutionary shift by combining phylogenetic inference with functional and biochemical/ biophysical techniques to resurrect AncSR1 and AncSR2 and experimentally characterize them; 2) Identify the historical mutations that switched DNA specificity and characterize the mechanisms by which they did so, using targeted genetic manipulations and experimental analysis in ancestral backgrounds; 3) Identify permissive mutations that were required for AncSR1 to tolerate the mutations that shifted its DNA recognition and determine the mechanisms for their effects; and 4) Develop a new high-throughput method to identify the functional effects and interactions of all historical mutations between AncSR1 and AncSR2. Our experiments will establish a complete mechanistic account for the evolution of novel TF specificity, linking historical genetic changes to shifts in protein function and biochemistry that generated a new gene regulatory system. This complete causal chain will elucidate how the biophysical architecture of extant proteins evolved and how that architecture structured the evolutionary genetic process. As the first-of-its-kind case study of the mechanistic evolution of TF function, this project will establish a methodological exemplar for future studies. Because the architecture of SR binding to DNA is classical, our work will establish baseline knowledge of evolutionary processes that is likely to apply to other TF families. The resulting structure-function knowledge will facilitate efforts to engineer TFs with new DNA-binding specificities in synthetic biology and biomedicine.